The long term goal of this research is to develop a pharmacological treatment for cocaine abuse. Specifically, this proposal will examine the potential of a thermostable mutant of bacterial cocaine esterase to be used as a therapy for cocaine abuse. Cocaine abuse is a widespread problem in the United States. Chronic cocaine use leads to severe physical and social consequences yet currently there is no specific FDA-approved pharmacotherpay for cocaine abuse. This research focuses on the bacterial protein cocaine esterase (CocE), isolated from a Rhodococcus bacterium that lives in the 180C soil surrounding the coca plant. CocE rapidly hydrolyzes cocaine significantly faster than similar enzymes that are in development for cocaine abuse and toxicity. Previous work done by this lab has shown that small doses of cocE are able to effectively block cocaine-induced lethality, convulsions, seizure, as well as increases in blood pressure, heart rate and QT interval. Although the wild type form has an extremely short half-life at 370C in vitro (13 minutes), a new set of mutations, L169K/G173Q, give CocE an in vitro half life of 72 hours. The extended half-life and long duration of action of this mutant makes it a better candidate for an abuse therapeutic than wild-type CocE. Experience with wild-type CocE as well as other cocaine hydrolyzing molecules uniquley positions this group to advance the development of this thermostable mutant of CocE towards a therapy for cocaine abuse. Based on the hypothesis that the long acting mutation of bacterial CocE (L169K/G173Q) may be an effective potential treatment for cocaine addiction, two specific aims are proposed: 1) Determine the efficacy of L169K/G173Q CocE against the lethal and reinforcing effects of cocaine in vivo. The potency and duration of action of L169K/G173Q CocE and wild type cocE against cocaine-induced lethality in NIH Swiss mice will be assessed. The effects of L169K/G173Q CocE on the reinforcing properties of cocaine will be assessed in a rat cocaine self-administration model. 2) Determine the stability and activity of L169K/G173Q CocE in vivo. Enzyme stability will be examined by analyzing the plasma half-life, in vivo rates of catalysis, ex vivo activity and immunogenicity of L169K/G173Q and wild-type CocE. We hope to develop a thermostable mutant bacterial cocaine esterase into a pharmacotherapy for cocaine abuse, a widespread social problem for which there is currently no specific FDA approved treatment. Thermostable cocaine esterase rapidly converts cocaine into inactive products, therefore eliminating or greatly reducing the strong psychotropic and physiological effects of cocaine.